Page 154 - Haematologica May 2020

B. Depreter et al.
vs. 10%) (Figure 4A). Moreover, TARP-TCR CTL were also capable of killing leukemic cells from de novo adult AML patients (n=5) (Figure 4E). Lysis ranged between 12% and 68%, and borderline correlated to TARP tran- script levels (Spearman's coefficient 0.82, P=0.089).
Discussion
We demonstrated increased TARP expression in AML LSC (CD34+CD38–) and blasts (CD34+CD38+) from pri- mary patients compared to their normal counterparts as well as AML cell lines. We also showed that TARP protein is expressed in primary AML leukemic cells and are ade- quately presented on HLA molecules, which makes the cells targetable for immunotherapy.
TARP expression has only been investigated in prostate tissue and androgen-sensitive prostate adenocarcinoma and breast adenocarcinoma,42,43,48 next to a single report on salivary adenoid cystic carcinoma.49 We found that TARP expression was significantly (P<0.001) higher in FLT3- ITD compared to FLT3 WT pedAML patients at diagnosis, whereas no significant difference was observed in adult AML. Importantly, enormous differences in the genomic landscape in adult compared to pedAML were shown,50,51 potentially explaining some of the differential associations observed in our cohorts. The association between TARP expression and a poor prognosis is in agreement with a recent report, investigating the association between tran- script expression and clinical outcome in pedAML, rank- ing TARP within the top genes significantly associated with a detrimental outcome.52 To shed light on the link between FLT3-ITD and TARP, mRNA sequencing of the transgenic OE and KD cell lines compared to their WT cell line is ongoing. As it was recently shown that the FLT3-ITD regions encode immunogenic, HLA-presented neo-epitopes,53 the benefit of CTL therapy targeting both leukemogenic molecules in pedAML could be of great interest. On the other hand, CBF leukemias, representing a favorable cytogenetic subgroup,2,8 were only present (P<0.01) in the TARP-low group for both pediatric and adult patients. AML cell lines derived from pediatric cases (MV4;11, THP-1) and LSC-enriched cell lines (Kg-1a, HNT-34), showed the highest TARP levels, confirming a relation between TARP, the LSC compartment and pedAML, although also HL-60 (adult, CD34–) showed high expression. Whether TARP remains differentially expressed within LSC outside the predominant CD34+CD38– compartment, as it does within CD34– AML,15,54 needs to be explored further. In addition, we showed that transcripts differ from those in solid tumors and are derived from both the TRGC1 and TRGC2 coding regions. Sequencing analysis indicated the presence of a second, AML-exclusive, TARP transcript encoding TRGC2 instead of TRGC1.
TARP protein expression was in agreement with tran- script levels, showing a 15-25 kDa fragment in AML cell lines. In breast and prostate adenocarcinoma, TARP had previously been defined as a 7 kDa protein,42,48 although also a 9 kDa fragment was reported in MCF-7.42,48 Fritzsche et al. detected protein sizes in prostate carcino- ma of 20-25 kDa,55 comparable to our findings, whereas Yue et al. reported a 15 kDa protein.49 Besides its size, the subcellular localization of TARP in AML needs to be refined. qPCR analysis revealed cytoplasmic localization,
and confocal microscopy showed sporadic ER overlap, in contrast to previously reported mitochondrial co-localiza- tion.43 We observed an enrichment of TARP at the cells’ protrusions in Kg-1a and sorted leukemic cells. Protrusions are kinetic cytoskeletal abnormalities formed during chemokine-induced cell migration, e.g. homing of CD34+ HSC towards the bone marrow niche.56 The pres- ence of molecular abnormalities in CD34+ progenitor cells was shown to increase protrusion formation.57 Indeed, LSC were reported to compete with HSC for endosteal niche engraftment, where they are protected from chemotherapy-induced apoptosis.12,58 Whether TARP interferes in homing and chemoprotection of leukemic AML cells in the BM microenvironment needs to be elu- cidated. Although protein expression was readily up-reg- ulated in TARP transgenic cell lines, shRNA-mediated knockdown appeared to be less efficient. Possible expla- nations are the presence of escape mechanisms and alter- native translation pathways during silencing or a very high stability of the TARP protein, persisting in the cell for a long period of time.
To explore TARP as an immunotherapeutic target in AML, we evaluated the cytokine release and cytotoxic killing capacities of TARP-TCR transgenic CTL in vitro. TARP and HLA-A*0201 co-expressing cell lines were effi- ciently lysed, and although evaluated on a limited number of patients (n=5), TARP-TCR CTL were able to kill pri- mary leukemic cells with a borderline correlation to the TARP transcript expression. Interestingly, weaker respons- es were observed for the cognate TARP4-13 peptide, since the TCR is directed against the HLA-A*0201 enhanced affinity TARP(P5L)4-13 peptide. Moreover, pulsed T2 cells appeared to be more susceptible than AML cells. This finding is in agreement with previous data,47,59 and several reasons may account for this phenomenon. First, peptide processing, transport and/or MHC-I presentation may be disturbed in leukemic cells.60 Second, high and stable HLA- A*0201 expression is vital for triggering lytic responses, and transgenic expression might diminish during culture. Therefore, we cannot exclude the possibility that HLA- A*0201-mediated TARP presentation within the trans- genic OCI-AML3 cell line had diminished during long- term culture. Third, competition between transgenic and endogenous MHC-I molecules might block HLA-A*0201- guided peptide presentation. Indeed, the TARP-TCR was shown to suffer from low MHC-I avidity compared to for- eign epitope-directed TCR.61 Cloning the TARP4-13-TCR sequence into a retroviral construct enabled higher trans- duction efficiencies and the generation of mock CTL to correct non-TARP mediated lysis, which are lacking in previous reports.37,49 As promoters driving TCR expression differed between constructs, and functional activity is known to correlate with TCR cell-surface expression,62 it was no surprised that different killing rates between LV and RV transduced CTL were osberved. In addition, intrinsic reactivity, HLA status and endogenous TCR repertoire of each donor as such might have an impact.62 In addition, comparing reactivity by effectors from an allo- geneic versus autologous setting will be implemented in future experiments.
In conclusion, we showed that TARP is highly expressed in AML leukemic cells, including the CD34+CD38– LSC compartment, while absent in normal counterparts. Moreover, TARP expression was associated with FLT3-ITD in pedAML. We provide in vitro evidence that
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